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Rapid detection of SARS-CoV-2 RNA using a one-step fast multiplex RT-PCR coupled to lateral flow immunoassay

BMC Infectious Diseases volume 24, Article number: 1417 (2024) Cite this article

Abstract

Background

The COVID-19 has put emphasis on pivotal needs for diagnosis and surveillance worldwide, with the subsequent shortage of diagnostic reagents and kits. Therefore, it has become strategic for the countries to access diagnostics, expand testing capacity, and develop their own diagnostic capabilities and alternative rapid accurate nucleic acid diagnostics that are at lower costs. Here, we propose a visual SARS-CoV-2 detection using a one-step fast multiplex reverse transcription-PCR (RT-PCR) amplification coupled to lateral flow immunoassay detection on a PCRD device (Abingdon Health, UK).

Methods

We developed various simplex fast-PCRs for screening sets of primer pairs newly designed or selected from literature or from validated WHO diagnostics, targeting S, N, E, RdRp or ORF1ab genes. We retained primers showing specific and stable amplification to assess for their suitability for detection on PCRD. Thus, fast RT-PCR amplifications were performed using the retained primers. They were doubly labeled with Fam and Biotin or Dig and Biotin to allow visual detection of the labeled amplicons on the lateral flow immunoassay PCR Detection (PCRD) device, looking at lack of interaction of the labeled primers (or primer dimers) with the test-lines in negative or no RNA controls. We set up all the assays using RNAs isolated from patients’ nasopharyngeal swabs. We used two simplex assays, targeting two different viral genomic regions (N and E) and showing specific detection on PCRD, to set up a one-step fast multiplex RT-PCR assay (where both differently labeled primer pairs were engaged) coupled to amplicons’ detection on a PCRD device. We evaluated this novel assay on 50 SARS-CoV-2 positive and 50 SARS-CoV-2 negative samples and compared its performance to the results of the quantitative RT-PCR (RT-qPCR) assays used for diagnosing the patients, here considered as the standard tests.

Results

The new assay achieved a sensitivity of 88% (44/50) and a specificity of 98% (49/50). All patients who presented Ct values lower than 33 were positive for our assay. Except for one patient, those with Ct values above 33 returned negative results.

Conclusion

Our results have brought proof of principle on the usefulness of the one-step fast multiplex RT- PCR assay coupled to PCRD as a new assay for specific, sensitive, and rapid detection of SARS-CoV-2 without requiring costly laboratory equipment, and thus, at reduced costs in a format prone to be deployed when resources are limited. This assay offers a viable alternative for COVID-19 diagnosis or screening at points of need.

Peer Review reports

Introduction

The COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is an emerging and highly infectious disease that has rapidly spread worldwide and become a public health emergency. The pandemic has placed considerable pressure on global health systems, and has notably highlighted the importance of diagnostics while exposing shortage in diagnostic reagents and kits. Thus, it is strategic for the countries to enhance access to diagnosis and acquire the capacity to face such needs now and during future threats. Early detection of the disease was a key step in controlling its spread. The molecular diagnosis of COVID-19 is based on the amplification of viral genetic material. The World Health Organization (WHO) has designated reverse transcription and real-time quantitative PCR (RT-qPCR) as the gold standard diagnosis technique using a selection of protocols aiming at the amplification of one or more viral genome targets. Different protocols have been developed by different institutions in the United States, Germany, France, China, Hong Kong, Japan and Thailand, targeting different viral genomic regions (WHO, 2020). For example, the United States Center for Disease Control (US-CDC) protocol used three nucleocapsid gene targets (N1, N2 and N3). The protocol developed by German Consiliary Laboratory for Coronaviruses hosted at the Charité in Berlin (Charité/Berlin) used first line screening with the envelope (E) gene assay followed by a confirmatory assay using the RNA-dependent RNA polymerase (RdRp) gene [1]. The Hong Kong University, China protocol used two simplex assays targeting the nucleoprotein (N) gene and the Open reading frame ORF1ab [1]. The protocols developed by the Institiut Pasteur of Paris (IP2 and IP4) targeted two RdRp regions [1]. The China CDC protocol targeted the N gene and ORF1ab [1]. The one from Thailand was developed by the Department of Medical Sciences, Ministry of Public Health, and targeted the N gene [1].

Multiplex RT-qPCR assays have also been developed, representing an interesting advancement allowing simultaneous detection of two or more targets [2, 3]. Another advancement in RT-qPCR was the development of an RNA extraction-free assay capable of detecting SARS-CoV-2 directly from patient nasopharyngeal swabs, maintaining adequate specificity and sensitivity [4]. Nevertheless, the RT-qPCR assays have been deployed in centralized diagnosis centers, and their major drawback is the need for relatively complex and expensive equipment and highly qualified personnel. This limited their use as a diagnostic tool in settings lacking adequate infrastructure and equipped laboratories [5]. Therefore, developing simpler molecular assays will improve testing approach and allow for the development of diagnosis algorithms, thus, overcoming equipment and resource challenges. In addition, given the worldwide pressure to access reagents during the pandemics, alternative solutions should be considered to address supply shortages, to lower diagnostic costs and thus, to expand testing capacity, and to better prepare for future pandemics. Testing is a cornerstone to fighting pandemics, for surveillance, and during likely resurgence events. Several assays have been developed to meet this need. They mainly include isothermal assays like loop-mediated isothermal amplification (LAMP), a technology that allows nucleic acid amplification at a constant temperature of 60 °C to 65 °C [6]. It uses a DNA polymerase with high strand displacement activity and four to six pairs of primers [7]. An additional reverse transcription step was combined, in a single tube, with the LAMP amplification technique, minimizing the reaction time to approximately 30 min [8]. RT-LAMP is less expensive, simpler, and faster to detect DNA than PCR, and it does not require the use of a thermocycler or expensive reagents [8]. It has been used for the direct detection of SARS-CoV-2 RNA targeting the N, ORF1ab, and RNA polymerase genes [8, 9]. As a simple and rapid technology, RT-LAMP can be coupled with various assay types for visualizing amplified products; the most commonly used ones include visualizing amplified products with the naked eye either through a color indicator change [8, 9] or on lateral flow strips [10]. Although the performance of LAMP is comparable to that of RT-qPCR for SARS-CoV-2 detection [11], it is limited by the complexity of primer design [12] and the generation of false-positive results [13]. Moreover, multiplexing is complex due to the number of primers needed for a single reaction.

Other widely used isothermal technologies include recombinase-based amplification that includes recombinase polymerase amplification (RPA) and the similar recombinase-aided amplification (RAA). It has also been used for SARS-CoV-2 detection. It represents a faster, simpler, and less expensive alternative to RT-qPCR. It allows amplification at 37–42 °C using only a pair of primers and a mix of enzymes (including recombinase) that eliminate the need for instruments such as thermocyclers [14]. This technology simplifies the testing process and allows SARS-CoV-2 detection in resource-limited environments. RPA/RAA offers various strategies for detecting amplified products, which can be either real-time using labeled probes generating visually detectable signals in 15 min (owing to fluorescent label) [15] or lateral flow strips in 5 min (owing to 6-FITC/biotin labels) [16]. However, longer primer sequences as well as lower primer binding temperatures in recombinase-based amplification reactions result in the formation of primer-dimers that may produce false positive signals [17].

CRISPR-Cas technology has recently been employed as another alternative for highly sensitive and specific detection [18, 19]. Several CRISPR-based approaches such as SHERLOCK [20], DETECTR [21], ENHANCE [22], iSCAN [23], FELUDA [24], and AIOD-CRISPR [25], have been developed for accurate and portable diagnosis of COVID-19 from various types of samples. Nevertheless, CRISPR-based approaches are always associated with a pre-amplification step, which makes them time consuming and prone to cross-contamination.

Microfluidic techniques have also been developed for COVID-19 diagnosis, as they are ideal for point-of-care applications due to their simple structure, compact size, and cost-effectiveness [26]. They represent platforms that integrate systems for RNA extraction, RT-PCR, and optical readers or lateral flow chromatography [26]. However, these novel technologies have limited sensitivity compared to traditional laboratory-based methods, potentially leading to false negatives, especially in samples with low viral loads [27].

Sequencing technologies have also played a pivotal role during the pandemic. Using next-generation sequencing (NGS) techniques, researchers successfully obtained the first SARS-CoV-2 genome in under a month following the identification of the disease. Since then, numerous sequencing-based diagnostics have been developed and utilized [19]. However, their application for SARS-CoV-2 detection remains limited due to their high throughput nature and the complexity of data analysis [19].

Despite the important number of novel technologies developed for pathogen detection, PCR remains the gold standard molecular diagnostic technique for infectious diseases. During the last decade, it has evolved to achieve better performance in terms of sensitivity, cost, and duration [28]. The introduction of DNA polymerases with improved processivity, drastically reduced the duration of a reaction and made fast-PCR an interesting alternative for rapid pathogen detection [29]. Thermocyclers, which are necessary for PCR, are becoming increasingly affordable and are currently considered basic equipment for infectious disease diagnosis laboratories. Furthermore, portable formats are now commercially available, enabling the creation of highly useful mobile laboratories to be closer to the patients or for field investigation notably to promptly respond to health emergencies that require immediate intervention.

During the COVID-19, one of the most used technique for disease diagnosis was based on lateral flow immunoassays for the detection of SARS-CoV2 antigens or human antibodies (anti-SARS-Cov2-IgG and anti-SARS-Cov2-IgM) thanks to their accessibility, feasibility and affordability [30, 31]. Commercially available generic lateral flow immunoassays have enabled the accurate detection of nucleic acids from a range of pathogens [29, 32, 33]. These accessible assays proved to be user-friendly, cost effective making them strong candidates for decentralized diagnosis of infectious diseases and control strategies [34].

In this study, we aimed to address needs for alternative, rapid and less expensive approach to COVID-19 diagnosis by developing a one-step fast multiplex reverse transcription-PCR (RT-PCR) coupled to visual detection on a lateral flow immunoassay PCR Detection (PCRD) device. This study provides a proof of principle for the potential of these new assays for rapid amplification and detection of viral RNA, and supports the feasibility of adapting them into a mobile version.

Materials and methods

Ethical statement

The study is approved by the Biomedical Ethics Committee of the Institut Pasteur de Tunis (Ref: 2020/21/I/LR16IPT) in accordance with the Declaration of Helsinki.

Human samples

Human samples used in this study correspond to nasopharyngeal swabs collected from anonymized patients suspected of COVID-19 disease and referred for routine diagnosis to the Clinical Virology Laboratory of the Institut Pasteur de Tunis. Patients were enrolled between November 2020 and January 2021. They include 50 SARS-CoV-2-positive (CoV+) and 52 SARS-CoV-2-negative (CoV-) for COVID-19, as shown by the RT-qPCR (Table 1). RNAs were extracted using QIAamp Viral RNA Mini Kit (Qiagen, USA). Thirty additional RNAs extracted from 15 CoV+ (C+) and 15 CoV- (C-) were used for primers screening. For these RNAs, the cDNAs was synthesized using M-MuLV reverse transcriptase (GeneON-Bioscience, Germany). The cDNA synthesis included first annealing step where 10 µM of OligodT(23) were incubated with the RNA in a final volume of 8 µl for 10 min at 70 °C and then for 5 min at 4 °C. During the second step, the cDNA synthesis reaction itself was performed by mixing the OligodT-RNA complex with 1X reaction buffer, 0.5 mM of dNTPs and 200 U of M-MuLV reverse transcriptase and incubating the mix at 42 °C for 1 h and at 65 °C for 10 min. Synthesis was then checked by amplifying the human beta-globin gene (β-globin) as described in [35].

Table 1 Results of the evaluation of the one-step fast multiplex RT-PCR/PCRD assays on clinical samples
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Targets selection and primers design

We performed a bibliography search to identify the most used genes in published protocols for the diagnosis of COVID-19. The primers used in this study were selected from the bibliography, from protocols approved by the WHO, or were manually designed in our laboratory. We designed a total of 13 primer pairs targeting genes encoding the S protein (N = 6 pairs), the N protein (N = 2 pairs), the E protein (N = 2 pairs), and the open reading frame ORF1ab (N = 3 pairs). We also selected five other primer pairs published and validated by the WHO for RT-qPCR protocols. They correspond to primers described in the protocols of the Institut Pasteur of Paris (IP2 and IP4) targeting the RdRp gene, the protocol described by the US-CDC targeting the N gene (US-CDC-N2), the protocol described by the China-CDC targeting the ORF1ab region and finally the protocol described by China Hong Kong University (China-HKU) targeting ORF1ab. Three additional primer sets were selected from the literature, they target the E gene [36], the RdRp gene [37] and ORF1ab [38]. We selected/designed primers that did not show secondary structures or dimers based on NetPrimer software analysis (NetPrimer :: PREMIER Biosoft) to avoid background noise during the PCRD readout. The primer screening, thus, included a total of 23 primer pairs combinations targeting 5 regions (E, N, S, RdRp and ORF1ab) of the viral genome (Table 2). Strategy used for assays set up is described in the Supplementary Fig. 1.

Table 2 Targets and primers used for the set-up of the assays
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Fast simplex PCR assays for target screening

Screening assays were undertaken on cDNAs synthesized from RNAs extracted from nasopharyngeal swabs of CoV + and CoV- patients using M-MuLV reverse transcriptase (GeneON-Bioscience, Germany) as described above. Fast simplex (engaging one primer pair) PCR assays were performed using SolisFAST Master Mix (Solis Biodyne, Estonia) containing a fast hot-start and fast extension DNA polymerase. The reaction was performed in 20 µl containing a ready-to-use 1X Master mix, 0.5 µM for each forward and reverse primer and template cDNA. The reactions were run for 45 min using the following fast PCR program: initial denaturation at 98 °C for 2 min followed by 35 cycles of denaturation at 98 °C for 10 s, annealing for 30 s at the appropriate temperature for each primer pair tested, and extension at 72 °C for 30 s. The amplification products were then visualized on a 2% agarose gel. The selection criteria of the primer pairs were mainly based on specificity, stability, and reproducibility of the amplification results.

PCRD detection assays

The primer pairs that gave specific and reproducible results, when analyzed by electrophoresis on agarose gels, were selected to be labeled for the purpose of their use for lateral flow immunoassay detection using PCRD cassettes as recommended by the manufacturer (Abingdon Health, UK). PCRD lateral flow detection is a two-test-line sandwich immuno-chromatography- based assay that relies on fluorescein (Fam)/Biotin and digoxigenin (Dig)/Biotin labeled primers used for the PCR assays. Six µl of the PCR products were diluted in 84 µl dilution buffer (Abingdon Health, UK), 75 µl of which were then transferred to the sample pad of the PCRD cassette as recommended by the manufacturer. During the flow migration, labeled amplicons are captured by the antibodies (anti-Biotin) immobilized at the test-lines to form colored complexes and therefore become a visible line. The result was then read with the naked eye after 5, 10 and 15 min of flow migration, and pictures were taken for our records.

One-step fast multiplex RT-PCR assay and PCRD detection optimization and set up

The selected primer pairs in the simplex assays (Table 3) were used to set up a one-step fast multiplex RT-PCR (that target in the same tube two different viral genome regions) coupled to PCRD detection. The one-step fast multiplex RT-PCR assays were performed using the ready-to use Palm PCR Express One-Step RT-PCR Kit I (Ahram Biosystems, Korea). The PCR mixture (20 µl) contained 1X Palm PCR Master Mix (0.8 U Taq polymerase, 50 U reverse transcriptase, 2.5 mM MgCl2 and 0.2 mM each dNTPs) and two differently labeled primer pairs. Three different primers concentrations were tested (0.3 µM, 0.4 µM and 0.5 µM) to select most optimal one. The one-step fast multiplex RT-PCR assays were performed in a thermocycler using the following cycling parameters: 30 min of reverse transcription at 50 °C, an initial denaturation at 95 °C for 1 min followed by 35 cycles consisting of 95 °C for 5 s and an annealing/extension step for 10 s. Two annealing temperatures, 56 °C and 57 °C, were tested to set up this assay. The amplification products were then visualized on a 2% agarose gel upon electrophoresis and on a PCRD cassette, as described above in Sect. 5.

Performance evaluation of the fast multiplex RT-PCR coupled to the PCRD detection assays using the optimized protocols

The optimized fast multiplex RT-PCR assay, using the selected primers pairs, was tested on 50 CoV + and 52 CoV- RNAs as defined by the RT-qPCR standard technique (Table 1). In parallel, RT-fast PCR targeting the human β-globin gene was performed using the Palm PCR Express One-step RT PCR Kit I in order to verify RNA integrity. The RT-PCR protocol used for β-Globin is the same as that used for the fast multiplex RT-PCR described in the previous section (Sect. 6). We used GH20/PCO4 primers with a concentration of 0.16 µM and the annealing temperature was set to 57 °C. The multiplex and β-Globin RT-PCRs were run in parallel, at the same time and in the same thermocycler.

Sensitivity and specificity were computed to evaluate performance of the new assay using RT-qPCR as a gold standard technique.

Results

The primers and targets were selected through fast simplex PCR and PCRD detection assays

The already described primers and the new ones designed (Table 2) were tested in fast simplex PCR assays against the selection criteria. Twelve primer pairs showed specific and reproducible amplification in the agarose gels. These primer pairs were labeled for PCRD detection. However, save for two pairs, all the tested primer pairs showed background noise with the negative and no template controls when PCR products were visualized in the PCRD (Fig. 1).

Fig. 1
figure 1

PCRD lateral flow detection of some rejected targets due to background noise (red arrows) in the negative (C-) and No template control (NTC) samples. C+: Positive control, Black arrows: Expected result

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The two remaining primer pairs showed specific and stable results with no artefacts when negative samples (CoV-) and no template controls (NTC) were tested (Fig. 2). They correspond to primers used to amplify the N gene (US-CDC-N2) and those amplifying the E gene [36].

Fig. 2
figure 2

Simplex fast RT-PCR assays targeting the N and E genes visualized on (a) 2% agarose gels and (b) PCRD. Test-line 1 detects the Dig/biotin-labeled E target amplicon. Test-line 2 detects the Fam/biotin-labeled N target amplicon. C: Control line for flow migration. MW: 100 bp Molecular weight. CoV+: SARS-CoV-2 positive samples. CoV-: SARS-CoV-2 negative samples. NTC: No Template Control. The black arrows show positive results

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To allow PCRD lateral flow detection, these primers were differently labeled: N-F-Fam/R-Biotin and E-F-Dig/R-Biotin, respectively (Table 3). The amplicons were first visualized on agarose gels, followed by PCRD lateral flow detection (Fig. 2). On the PCRD, the N gene amplicon is captured on test-line 1, while the E gene amplicon is captured on the test-line 2 (Fig. 2).

Table 3 Selected primers for fast multiplex RT-PCR and PCRD detection and corresponding labels
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A one-step fast multiplex RT-PCR and a PCRD detection were set using the primer pairs selected

The 2 selected primer pairs were engaged in a one-step fast multiplex RT-PCR where they were added to the same reaction mixture. The reaction conditions were established by varying primers concentrations (Fig. 3a) and melting temperatures (Fig. 3b) in an attempt to balance band intensities as band of the E amplicon was more intense than the N one in both agarose gel and PCRD. In the retained protocol, the primers final concentrations were set to 0.5 µM each and the Tm at 57 °C. The one-step fast multiplex RT-PCR requires 1 h 15 min including 30 min for reverse transcription (RT) and 45 min for PCR. The amplification products were visualized on agarose gel and PCRD lateral flow. The PCRD lateral flow detection showed that there were no differences between 5, 10, and 15 min of flow migrations (Fig. 3c). Therefore, the results were read, and the corresponding pictures were taken after 5 min of flow migration.

Fig. 3
figure 3

Optimization of the fast multiplex RT-PCR targeting N and E genes. The tested conditions are (a) Primers’ concentrations, (b) Tm and (c) Time for PCRD detection. [E] and [N] are the concentrations tested for each primer pair. C+: [2642, 2911, 2866]. NTC: No template control

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The performance of the established one-step fast multiplex RT-PCR coupled to the PCRD detection was evaluated using nasopharyngeal samples

The retained amplification and detection protocols were applied to a collection of 102 RNAs extracted from patient’s nasopharyngeal swabs to evaluate the performance of the one-step fast multiplex RT-PCR and the PCRD detection assays. The status of the collected RNAs was confirmed by RT-qPCR at time of sampling: 50 were positive (CoV+) and 52 were negative (CoV-). The RNA integrity, at time of testing the novel assay, was verified by one-step fast RT-PCR amplification of the human β-globin gene. In case of two samples (52 and 148), the fast RT-PCR for β-globin was negative (Table 1). Therefore, these two samples were excluded from the study. We tested the remaining 100 RNAs and considered the assay positive if, at least one of the test-lines showed a positive result. If both test-lines showed negative results, the assay was considered negative (Table 1). Our one-step fast multiplex RT-PCR assay coupled to PCRD detection achieved a sensitivity of 88% (44/50) and a specificity of 98% (49/50).

For some samples, the N target showed ambiguous results on the agarose gel, as the amplicons were easily mistaken for primers dimers and/or primers excess because of their small size (67 bp). This ambiguity is resolved by the PCRD detection, as primer dimers and/or excess primers detected on the agarose gel were not visible on test line 2, where the N gene amplicons are captured. Only positive samples (CoV+) showed positive results on the PCRD (Fig. 4). The PCRD ensures a higher confidence level in interpreting positive results as it was able to distinguish between true target amplification and non-specific artifacts, such as primer dimers or excess primers. This precision minimizes the likelihood of false positives or misinterpretation.

Fig. 4
figure 4

Results of the one-step fast multiplex RT-PCR on CoV + and CoV- samples. The N target produced ambiguous results in agarose gel, which were resolved by PCRD detection. CoV-: Negative samples, CoV+: Positive samples, MW: 100 bp Molecular weight, NTC: No template control, Black arrow: Positive test-line

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Additionally, we noticed that this one-step fast multiplex RT-PCR/PCRD assay is able to detect all positive samples having Ct values lower than 33. Samples with Ct values above 33 returned negative results, except for one sample (285), which had Ct values of 37/38, but gave positive results for both targets tested. Concordance and discrepancies between RT-qPCR and the one-step fast multiplex RT-PCR/PCRD assays, based on Ct values, are summarized in Table 4.

Table 4 RT-qPCR and multiplex fast PCR/PCRD assay results across different patient groups
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Discussion

In the context of strengthening local and international diagnostic facilities and technical capacity for pathogen detection during pandemics, epidemics or outbreaks, our study aims to contribute to the development of simple and rapid assays that do not require the use of sophisticated equipment and can be implemented in minimally equipped healthcare centers. Indeed, the emergence of infectious diseases, such as COVID-19, has stressed the importance and necessity of rapid and effective diagnosis for detecting pathogens and controlling their spread. COVID-19 represented an international public health emergency due to its pathogenesis and rapid spread. Simple, rapid, sensitive, specific, and equipment-free diagnostic assays were essential to meet the needs of epidemic control strategies and thus, limit the spread of the disease. While different approaches have been used for disease diagnosis based on serology and computing tomography imaging, the WHO recommended real-time reverse transcription polymerase chain reaction (RT-qPCR) protocols as the reference assays [1]. The classical RT-qPCR is a sensitive and accurate technology but has several disadvantages, including the high cost of kits, the prolonged time to result delivery, the need for trained personnel, and the requirement for a suitable and costly piece of equipment that is usually missing in low-resource setting areas [39]. The development of simple, rapid, accurate, and field-applicable assays for detecting SARS-CoV-2 and future emerging pathogens remains a pressing need. Various molecular diagnostic alternatives, such as RT-LAMP [10], RT-RPA [16], and next generation diagnostics based on the CRISPR-Cas systems [21, 23, 24] and microfluidics [27], have been developed for detecting the SARS-CoV-2 virus. These diagnostics allow for rapid, specific, and effective detection under isothermal conditions, therefore, eliminating the need for sophisticated equipment. They also offer the possibility to miniaturize the devices used and shorten testing cycles [19, 26]. However, despite their advantages, these techniques have drawbacks, namely complex primer design and optimization of reaction conditions in case of LAMP [5], the risk of false positives with RPA [40], and multiple workflow steps for the CRISPR Cas systems [41]. In addition, for microfluidics, there remains a significant gap between cutting-edge laboratory findings and practical implementation. The challenge stems from the complexities of manufacturing and scaling these devices. Technologies used in developing microfluidic chips, require expensive infrastructure and the high cost makes these products inaccessible to many individuals [27, 42]. As PCR is still one of the most valuable technology used in different fields, including food security, forensics, research and biomedicine, in this study, we chose to develop PCR assays using newly designed primers or re-adapt the WHO and published real time PCRs into simpler PCR-based assays. The assay here reported is based on a one-step fast multiplex RT-PCR using a ready-to-use master mix and two pairs of already described primers that is coupled to lateral flow PCRD immunoassay detection. It is faster than the classical real time PCR. It is also easy to perform and read, and it obviates need for skilled operators and costly equipment mobilization that makes RT-qPCR only available in central laboratories and the ensuing transfer of samples to these central laboratories, which results in delays in result delivery. Indeed, for example, in Tunisia during the pandemic, real time PCR facilities and expert technicians were missing in different regions of the country. Therefore, in the beginning of the COVID-19, all the samples from most regions of the country were transferred to the Institut Pasteur de Tunis. Personnel, including MDs, researchers and lab technicians from different departments and labs of the institute, were called to join the COVID-19 testing team and were organized into three daily shifts, which led to postponing all other research and/or diagnosis activities. Therefore, one of the strategic actions for preparedness for future pandemics, epidemics or outbreaks must be the investment in laboratory infrastructure and diagnostic capabilities [43]. Here, we bring a proof of concept that the one-step fast multiplex RT-PCR coupled to PCRD detection could be a good alternative when there is a shortage in reagents or at points of need where real time PCR facilities are missing. In addition, this technology could be easily applied for the diagnosis of other pathogens [28]. Fast PCR and PCRD lateral flow technologies can be adapted to detect pathogens beyond SARS-CoV-2 due to their flexibility and scalability. This adaptability relies on the ability to select and/or design primers specific to DNA or RNA targets of various pathogens. These primers can be labeled with appropriate tags, making them compatible for PCRD detection. Moreover, multiple sets of primers can be labeled with distinct tags, enabling the simultaneous detection of several targets or pathogens in a single reaction. This approach could expand diagnostic capabilities to other infectious diseases, improve detection in resource-limited settings, and support surveillance of emerging pathogens. Furthermore, lateral flow immuno-chromatographic assay devices are commercially available. They represent a simple assay for the generic detection of nucleic acids without the need for specialized and costly equipment. Their use relies on the detection of dual-labeled amplicons. We chose primers’ labels according to the device used; like in this study, we used different labels associations, Fam/biotin and Dig/biotin for each primer pair, which target two different genomic viral regions, to be able to detect the amplicons on their respective test-line on the PCRD cassette. Compared to agarose gel electrophoresis, the PCRD assay is rapid (5 min), easy to use and read, and does not require extensive molecular biology expertise. Our study showed that the results observed in the agarose gel upon electrophoresis are concordant with those obtained with the PCRD analysis of the E target amplification. However, for the N target, where the amplicon is smaller in length (67 bp), the PCRD assay was able to resolve interpretation ambiguity of the read outs on agarose gels, as the amplicons were not distinguishable from excess primers and/or primer dimers upon electrophoresis.

The most commonly used genomic regions of SARS-CoV-2 for RT-qPCR diagnosis are highly conserved and/or highly expressed genes [44]. Notably, they include the ORF1ab regions, the genes encoding for nucleocapsid (N), envelope (E), spike protein (S), membrane protein (M), RNA-dependent RNA polymerase (RdRp), hemagglutinin-esterase (HE), and helicase genes. In this study, we considered most of the mentioned genes to develop the assay aimed for. We selected and designed our primers in the N, S, E, RdRp and ORF1ab viral genomic sequences. Our selection criteria for the primers were based, namely, on the stability and reproducibility of the fast-PCR as analyzed by the agarose gel electrophoresis, and then on the absence of background noise on the PCRD. The two targets that meet these criteria were the N (US-CDC-N2) and E genes [36]. The multiplexing of the amplification of these two targets within a same reaction was also successful, but we noticed that there was an imbalance in terms of band intensity on both the agarose gel and PCRD. The E amplicon band was more intense compared to the N one, despite our attempts to improve the reaction conditions. This could be due to a sort of competition between primers for the same mix of reagents [29, 45]. The GC content of the target (40,4% for E and 52,2% for N) could also lead to preferential denaturation, resulting in preferential amplification [45], or secondary structures within the genomes could induce differential accessibility of targets [45, 46]. Nevertheless, we were able to read PCRD results easily by the naked eye for both the E and N targets. The two targets showed the same sensitivity and specificity when tested in the multiplexed assay but gave discordant results for two patients: patient 47 was positive for the E target but negative for the N target, and patient 311 was negative for the E target but positive for the N target. For these patients, mutations in the priming sites could have occurred, leading to negative results. Or, SARS-CoV-2 gene dynamic leads to differential genes expression depending on the patient status and disease stage [47, 48]. The interest in using multiple targets is to define the infected patient’s status and minimize false negative results [1, 47]. However, our results showed that our assay achieved a sensitivity of 88%. Six patients who were positive according to RT-qPCR were negative with our assay. These patients had Ct values above 33, except for patient 285, who had Ct values of 37/38 for the two targets tested in RT-qPCR but showed positive results for the two targets tested in our one-step fast multiplex RT-PCR/PCRD. In this case, we propose two potential hypotheses. First, our assay is not sensitive enough and is able to detect only patients with a high viral load and a Ct below 33. The second hypothesis, given the fact that the RNAs in this study were extracted in 2021 (and our assays were performed in 2024), they may be subject to potential RNA degradation. It is known that long-term stability of the RNA constitutes a factor influencing the performance of the molecular assays, especially in case of samples with a low viral load. Indeed, it was demonstrated that RNA with a low viral load is more prone to a reduction in its RNA content than RNA with a high viral load [49]. Therefore, our one-step fast multiplex RT-PCR assay should be evaluated with freshly extracted RNAs and, at the same time when RT-qPCR is performed, to accurately determine their performances. Our assay achieved a specificity of 98%. Notably, Patient 139, who tested negative by RT-qPCR, was identified as positive with our assay. Even if we considered this case a false positive, the possibility of technical errors, such as pipetting inaccuracies during the RT-qPCR process, cannot be excluded.

The main limitation of the study is that we did not include the internal control (human β-globin) in our multiplex assay. We have attempted to minimize this drawback by setting in parallel this internal control on the same day and run. The one-step fast simplex PCR targeting the human β-globin was performed in a separate reaction as one cannot visualize more than 2 amplicons on the PCRD lateral flow device. Indeed, to our knowledge, immunoassay devices with 3 test-lines are not yet commercially available. However, lateral flow strip based on Single-stranded Tag Hybridization (STH) chromatographic Printed-Array Strip (PAS) method has recently been utilized as a simple and effective approach for the multiplex detection of pathogens, capable of identifying more than four targets simultaneously [50, 51]. For future diagnostics development and assays improvement, integrating all the targets (viral targets and internal human control) into a single reaction by adapting our immunoassay system to the STH chromatographic PAS assay would be highly advantageous. Furthermore, incorporating a rapid, simple, and equipment-free RNA extraction step into our new assay will streamline the workflow and enhance its accessibility in resource-limited settings. A recent study describing an RNA extraction-free RT-PCR assay demonstrated the feasibility of such an approach, achieving reliable detection of SARS-CoV-2 directly from clinical samples [4]. This highlights the potential of adapting similar strategies to improve the usability and efficiency of our assay.

Conclusion

The COVID-19 proved the relevance and importance of molecular diagnostics for disease control and transmission monitoring. It stressed the need for simple, fast and equipment- free assays as alternative solutions to bring testing closer to the patients. In this study, we brought the proof of concept that the one-step fast multiplex RT-PCR coupled to PCRD detection, here developed, is a good alternative for SARS-CoV-2 detection. It requires a conventional PCR thermocycler and PCRD devices, with result delivery within only 1 h and 20 min. Importantly, the assay developed using the ready to use kit for a portable RT-PCR machine can be easily adapted to point of care settings by using commercially available portable thermocyclers.

Data availability

Nucleotide sequences of the SARS-CoV-2 used for primers design are available in NCBI public databases and are available at the following URL: txid2697049[organism: exp] - Nucleotide - NCBI (nih.gov). All other data supporting the findings of this study are available within the paper. Primer sequences are provided in Table 2.

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Acknowledgements

The authors acknowledge all the Virology Lab staff who contributed to the process of the COVID-19 diagnosis including sampling, RNA extraction and RT-qPCR performing. We would like to thank Ahmed Sahbi Chakroun for his technical support and his advices on kits and reagent purchasing.

Funding

This research was funded by the Ministry of Higher Education and Research Tunisia through the programme “Projet de Recherche Fédéré” PRF-Lutte COVID, “Programme d’Encouragement des Jeunes Chercheurs” (21PEJC D5P15) and the Research laboratory contract program LR16IPT04, and by the Institut Pasteur de Tunis through the intramural program ”Projet Collaboratif Interne-PCI39”.

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  1. Insaf Bel Hadj Ali and Hejer Souguir Equal contributions.

Authors and Affiliations

  1. Laboratory of Molecular Epidemiology and Experimental Pathology, LR16IPT04, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, LR16IPT04, Tunisia

    Insaf Bel Hadj Ali, Hejer Souguir, Mouna Melliti, Mohamed Vall Taleb Mohamed, Monia Ardhaoui, Yusr Saadi Ben Aoun & Ikram Guizani

  2. Laboratory of Clinical Virology, WHO Regional Reference Laboratory for Poliomyelitis and Measles for the EMR, Institut Pasteur de Tunis, Tunis, Tunisia

    Kaouther Ayouni, Sondes Haddad-Boubaker & Henda Triki

  3. Laboratory of Virus, Host and Vectors, LR20 IPT02, Institut Pasteur de Tunis, University of Tunis El Manar, Tunis, Tunisia

    Kaouther Ayouni, Sondes Haddad-Boubaker & Henda Triki

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Contributions

IBA: Conceptualization and study design, writing of the original draft, data analysis, supervision, funding acquisition, project administration, writing, review and editing; HS: Funding acquisition, Study design, Investigation, data analysis, project administration; MM: Investigation, data analysis; MVT: Investigation; MA: Investigation; YSBA: Study design; KA: Investigation; SHB: Investigation, resources; HT: Funding Acquisition, resources; IG: Conceptualization, study design, funding acquisition, project administration, writing, review and editing. All authors read and approved the final manuscript.

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Correspondence to Insaf Bel Hadj Ali.

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The study is approved by the Biomedical Ethics Committee of the Institut Pasteur de Tunis under the reference Ref: 2020/21/I/LR16IPT in accordance with the Declaration of Helsinki. The need for consent to participate was waived by the Biomedical Ethics Committee of Institut Pasteur de Tunis. We used RNA retrospectively collected in the frame of the routine diagnosis of the COVID-19. Anonymous extracted viral RNA samples were transferred to our laboratory for assays development.

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Bel Hadj Ali, I., Souguir, H., Melliti, M. et al. Rapid detection of SARS-CoV-2 RNA using a one-step fast multiplex RT-PCR coupled to lateral flow immunoassay. BMC Infect Dis 24, 1417 (2024). https://doi.org/10.1186/s12879-024-10296-1

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